1. Technical Field
The present disclosure relates to fluorescence analysis and, more particularly, to methods of normalizing fluorescence analyzers according to specific wavelength characteristics thereof.
2. Background of Related Art
Filter fluorometry refers generally to the process of illuminating a sample and quantitating the resulting, detected fluorescence signal. A filter fluorometer, designed to perform fluorometry, employs one or more light sources, optical filters, and detectors to select the wavelength ranges of both the illuminating light and the light detected. Generally, these two kinds of wavelength ranges do not overlap to any appreciable extent, and the detection wavelengths are longer than the illumination wavelengths. Using a predetermined calibration curve, the detected signal is correlated to the amount of fluorescent component present in the sample. For example, filter fluorometry (hereafter, simply “fluorometry”) may be used to measure the concentration of certain electrolytes in blood plasma or serum by their effect on the fluorescence of a test device, e.g., a slide.
Currently, electrolyte detection via fluorometry involves the use of sensors which include reagents, e.g., electrolyte-specific fluoroionophores (for Na+ and K+ detection) or acridinium reagents (for Cl− detection). Initially, a slide containing only the appropriate sensor (e.g., the “dry” slide) is read by an analyzer designed to perform fluorometry on such slides, in order to measure an “early” fluorescence intensity. This “early” fluorescence may be an average “early” fluorescence intensity of multiple “early” fluorescence intensity measurements. Next, the sample to be tested is added to and allowed to interact with the slide. The slide, including the sample (e.g., the “wet” slide), is again read by the analyzer in order to measure a “late” fluorescence intensity. Similarly as above, this “late” fluorescence intensity may be an average of multiple “late” fluorescence intensity measurements. These measured values are then corrected in accordance with a fluorescence baseline and an analyzer response is calculated based on the ratio of the baseline-corrected “early” and “late” fluorescence intensity measurements. More specifically, the analyzer response is calculated according to:
                              AR          e                =                                            I                              late                ,                e                                      -                          I                              baseline                ,                e                                                                        I                              early                ,                e                                      -                          I                              baseline                ,                e                                                                        EQ        ⁢                                  ⁢        1            Where “AR” is the analyzer response for the given electrolyte, “e,” and wherein the “l” values are the measured fluorescence intensities (or averages thereof). Note that “early” fluorescence intensity measurement may also be obtained soon after sample addition.
The analyzer response “AR,” may then be used, in accordance with the slide's lot calibration, to determine the specific electrolyte's concentration in the sample under test. However, there are multiple sources of variability, e.g., analyzer-to-analyzer variability (wavelength variation in the fluorescence detection module, dispensed volume variation, incubation temperature variation, timing variation, etc.) and also sensor-to-sensor variability, that may produce different analyzer responses for the same sample. It is therefore necessary to correct, or “normalize” these analyzer responses in order to accurately determine the electrolyte concentration in a given sample.
Current methods of normalizing analyzer response values “AR” typically require running “known” samples on each analyzer to be normalized. More specifically, in one particular method of normalizing analyzer responses, a target analyzer response value for a given electrolyte, slide lot, and sample electrolyte concentration is first assigned. Then, using that given slide lot, the samples, which have different, but known electrolyte concentrations, are run on the analyzer to be normalized, in order to determine an uncorrected analyzer response for each sample. Multiple runs may be performed, indexed, and averaged with one another to determine an average uncorrected analyzer response for each sample. These uncorrected analyzer responses are then compared to the assigned target analyzer response values that correspond to the given electrolyte concentrations in the samples to determine a specific normalization factor, “se,c,” for each of the samples, e.g., for each given electrolyte concentration. This normalization factor, “se,c,” is used for all slide lots, although it is only calculated based upon a given slide lot, e.g., the given slide lot mentioned above. Next, the electrolyte concentration-specific normalization factors are averaged (or otherwise correlated) to yield an electrolyte-specific normalization factor applicable to all concentrations of that electrolyte. This normalization factor, “se,” in turn, is used to correct, or “normalize” the analyzer response, e.g., according to:
                              AR                      e            ,            corr                          =                              AR            e                                s            e                                              EQ        ⁢                                  ⁢        2            
This corrected, or “normalized” analyzer response, “ARe,corr,” in conjunction with the slide's lot calibration, is then used to determine the electrolyte concentration of a specific electrolyte in an “unknown” sample.
Although the above-described normalization process, and similar test-based normalization processes, correct for a number of sources of variability, e.g., dispensed volume variation, incubation temperature variation, and timing variation, such processes also introduce a number of additional sources of variability, including: accurate determination of the target analyzer response values, accuracy of the sample concentrations, and the applicability to all slide lots of a single normalization factor determined for one slide lot. Further, the above-described methods also require that multiple samples be run on each analyzer to be normalized in order to determine the normalization factor used to normalize the analyzer response Therefore, a need exists to develop a more efficient and more accurate method for normalizing analyzer response values in fluorometry.
It has been found that a major source of variability in fluorometry for electrolyte detection results from variation in wavelength characteristics of the filter fluorometer. Further, it has been found that accounting for these wavelength-based variations alone (and not considering the other sources of variation discussed above: dispensed volume variation, incubation temperature variation, and timing variation) yields better overall precision, e.g., less analyzer-to-analyzer variation, as compared to slide-based normalization methods, such as those described above. However, producing filters and source(s) (e.g., LED(s)) for use in the analyzers that are optimized to the required individual analyzer accuracy such that wavelength variation is reduced to insignificant levels would significantly increase the costs associated with the manufacture and qualification of such analyzers. It would therefore be desirable to provide a method of wavelength-based normalization for reducing analyzer-to-analyzer variation in fluorometry.